The important role in development of site-specific DNA methylation has become increasingly apparent in recent reports from several laboratories, including our own. This is particularly obvious in erythroid differentiation, the best studied example. The molecular mechanism by which DNA methylation exerts its influence is not understood. We wish to extend our earlier studies on DNA methylation in erythroid differentiation by purifying and characterizing the several DNA methyltransferases (MTases) of murine erythroleukemia cells (Friend or MEL cells), as well as those of mouse teratocarcinoma cells and mouse embryos. A novel affinity-labeling protocol for the characterization of the spectrum of DNA methyltransferases in any cell type will be developed. The effect of DNA structure and sequence on the in vitro methylation with our purified DNA MTases will be investigated in Z-DNA containing plasmids and SV40 DNA. The study of the role of our DNA MTases in the functioning of the DNA replication complex will describe the in vivo mode of action of these enzymes. The reciprocal relationship between differentiation and malignant cell behavior is well exemplified by the induction of differentiation in the usually malignant mouse embryonal carcinoma cells. There is reason to believe that DNA methylation plays a role here also and we will characterize the methylation status of the DNA and compare the DNA methyltransferases from the uninduced and induced states of these cells, using cloned viral DNA (MoMuLV) as substrate. We will also look for and study the occurrence of specific single-strand methylation sites in tissue DNA and relate their presence to the program of differentiation. A new sequencing method will be developed to detect strand and site-specific hemimethylation; two alternate enzymatic methods are also proposed. The puzzling and important question of the apparent heterogeneity of the MEL cell DNA methylases may not be solved by biochemical studies alone and will be examined by cloning the mouse DNA methyltransferase gene or genes, using in vivo and in vitro immunization and monoclonal antibodies to prepare the MTase cDNAs. An understanding of the gene organization of the methylases will lead to an investigaiton of the manner in which these genes are expressed in differentiating and other tissues. All these studies will help in defining the molecular mechanism of differentiation and malignancy and of the relationship between these two processes.